The present invention relates to nucleic acid sequences coding for a newly identified splice variant of human metabotropic glutamate receptor 5 (mGluR5). The novel human receptor may be expressed in host cells which may be used to screen for agonist, antagonist, and modulatory molecules that act on the novel human mGluR5. These molecules acting on the novel human mGluR can be used to modulate the activity of the novel human receptor for the treatment of neurological disorders and diseases.
The invention also relates to nucleic acids encoding such receptors, genetically modified cells containing such nucleic acids, methods of screening for compounds that bind to or modulate the activity of such receptors, and methods of use relating to all of the foregoing.
The following description provides a summary of information related to the background of the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to that invention.
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Glutamate produces its effects on central neurons by binding to and thereby activating cell surface receptors. These receptors have been subdivided into two major classes, the ionotropic and metabotropic glutamate receptors, based on the structural features of the receptor proteins, the means by which the receptors transduce signals into the cell, and pharmacological profiles.
The ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that, upon binding glutamate, open to allow the selective influx of certain monovalent and divalent cations, thereby depolarizing the cell membrane. In addition, certain iGluRs with relatively high calcium permeability can activate a variety of calcium-dependent intracellular processes. These receptors are multisubunit protein complexes that may be homomeric or heteromeric in nature. The various iGluR subunits all share common structural motifs, including a relatively large amino-terminal extracellular domain (ECD), followed by two transmembrane domains (TMD), a second smaller extracellular domain, and a third TMD, before terminating with an intracellular carboxy-terminal domain. Historically the iGluRs were first subdivided pharmacologically into three classes based on preferential activation by the agonists xcex1-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate (KA), and N-methyl-D-aspartate (NMDA). Later, molecular cloning studies coupled with additional pharmacological studies revealed a greater diversity of iGluRs, in that multiple subtypes of AMPA, KA and NMDA receptors are expressed in the mammalian CNS Hollman and Heinemann (1994), Ann. Rev. Neurosci. 17:31.
The metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors capable of activating a variety of intracellular second messenger systems following the binding of glutamate.
Activation of mGluRs in intact mammalian neurons can elicit one or more of the following responses: activation of phospholipase C, increases in phosphoinositide (PI) hydrolysis, intracellular calcium release, activation of phospholipase D, activation or inhibition of adenylyl cyclase, increases or decreases in the formation of cyclic adenosine monophosphate (cAMP), activation of guanylyl cyclase, increases in the formation of cyclic guanosine monophosphate (cGMP), activation of phospholipase A2, increases in arachidonic acid release, and increases or decreases in the activity of ion channels (e.g., voltage- and ligand-gated ion channels). Schoepp and Conn (1993), Trends Pharmacol. Sci. 14:13; Schoepp (1994), Neurochem. Int. 24:439; Pin and Duvoisin (1995), Neuropharmacology 34:1.
Thus far, eight distinct mGluR subtypes have been isolated via molecular cloning, and named mGluR1 to mGluR8 according to the order in which they were discovered. Nakanishi (1994), Neuron 13:1031; Pin and Duvoisin (1995), Neuropharmacology 34:1; Knopfel et al. (1995), J Med. Chem. 38:1417. Further diversity occurs through the expression of alternatively spliced forms of certain mGluR subtypes. Pin et al. (1992), Proc. Natl. Acad. Sci. USA 89:10331; Minakami et al. (1994), BBRC 199:1136; Joly et al. (1995), J Neurosci. 15:3970. All of the mGluRs are structurally similar, in that they are single subunit membrane proteins possessing a large amino-terminal ECD, followed by seven putative TMDs, and an intracellular carboxy-terminal domain of variable length.
The eight mGluRs have been subdivided into three groups based on amino acid sequence homologies, the second messenger systems they utilize, and pharmacological characteristics. Nakanishi (1994), Neuron 13:1031; Pin and Duvoisin (1995), Neuropharmacology 34:1; Knopfel et al. (1995), J Med. Chem. 38:1417. The amino acid homology between mGluRs within a given group is approximately 70%, but drops to about 40% between mGluRs in different groups. For mGluRs in the same group, this relatedness is roughly paralleled by similarities in signal transduction mechanisms and pharmacological characteristics.
The Group I mGluRs comprise mGluR1, mGluR5, and their alternatively spliced variants. The binding of agonists to these receptors results in the activation of phospholipase C and the subsequent mobilization of intracellular calcium. For example, Xenopus oocytes expressing recombinant mGluR1 receptors have been utilized to demonstrate this effect indirectly by electrophysiological means. Masu et al. (1991), Nature 349:760; Pin et al. (1992), Proc. Natl. Acad. Sci. USA 89:10331. Similar results were achieved with oocytes expressing recombinant mGluR5 receptors. Abe et al. (1992), J Biol. Chem. 267:13361; Minakami et al. (1994), BBRC 199:1136; Joly et al. (1995), J Neurosci. 15:3970. Alternatively, agonist activation of recombinant mGluR1 receptors expressed in Chinese hamster ovary (CHO) cells stimulated PI hydrolysis, cAMP formation, and arachidonic acid release as measured by standard biochemical assays. Aramori and Nakanishi (1992), Neuron 8:757. In comparison, activation of mGluR5 receptors expressed in CHO cells stimulated PI hydrolysis and subsequent intracellular calcium transients, but no stimulation of cAMP formation or arachidonic acid release was observed. Abe et al. (1992), J Biol. Chem. 267:13361. However, activation of mGluR5 receptors expressed in LLC-PK1 cells does result in increased cAMP formation as well as PI hydrolysis. Joly et al. (1995), J Neurosci. 15:3970. The agonist potency profile for Group I mGluRs is quisqualate greater than glutamate=ibotenate greater than (2S,1xe2x80x2S,2xe2x80x2S)-2-carboxycyclopropyl)glycine (L-CCG-I) greater than (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). Quisqualate is relatively selective for Group I receptors, as compared to Group II and Group III mGluRs, but it also potently activates ionotropic AMPA receptors. Pin and Duvoisin (1995), Neuropharmacology 34:1; Knopfel et al. (1995), J Med. Chem. 38:1417.
The Group II mGluRs include mGluR2 and mGluR3. Activation of these receptors as expressed in CHO cells inhibits adenlyl cyclase activity via the inhibitory G protein, Gi, in a pertussis toxin-sensitive fashion. Tanabe et al. (1992), Neuron 8:169; Tanabe et al. (1993), J. Neurosci. 13:1372. The agonist potency profile for Group II receptors is L-CCG-1 greater than glutamate greater than A CPD greater than ibotenate greater than quisqualate. Preliminary studies suggest that L-CCG-I and (2S,1xe2x80x2R,2xe2x80x2R, 3xe2x80x2R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) are both relatively selective agonists for the Group II receptors versus other mGluRs (Knopfel et al. (1995), J. Med. Chem. 38:1417), but DCG-IV does exhibit agonist activity at iGluRs as well (Ishida et al. (1993), Br. J. Pharmacol. 109:1169).
The Group III mGluRs include mGluR4, mGluR6, mGluR7 and mGluR8. Like the Group II receptors, these mGluRs are negatively coupled to adenylyl cyclase to inhibit intracellular cAMP accumulation in a pertussis toxin-sensitive fashion when expressed in CHO cells. Tanabe et al. (1993), J. Neurosci. 13:1372; Nakajima et al. (1993), J. Biol. Chem. 268:11868; Okamoto et al. (1994), J. Biol. Chem. 269: 1231; Duvoisin et al. (1995), J. Neurosci. 15:3075. As a group, their agonist potency profile is (S)-2-amino-4-phosphonobutyric acid (L-AP4) greater than glutamate greater than ACPD greater than quisqualate, but mGluR8 may differ slightly with glutamate being more potent than L-AP4. Knopfel et al. (1995), J. Med. Chem. 38:1417; Duvoisin et al. (1995), J. Neurosci. 15:3075. Both L-AP4 and (S)-serine-O-phosphate (L-SOP) are relatively selective agonists for the Group III receptors.
Finally, the eight mGluR subtypes have unique patterns of expression within the mammalian CNS that in many instances are overlapping. Masu et al. (1991), Nature 349:760; Martin et al. (1992), Neuron 9:259; Ohishi et al. (1993), Neurosci. 53:1009; Tanabe et al. (1993), J. Neurosci. 13:1372; Ohishi et al. (1994), Neuron 13:55; Abe et al. (1992), J. Biol. Chem. 267:13361; Nakajima et al. (1993), J. Biol. Chem. 268:11868; Okamoto et al. (1994), J. Biol. Chem. 269:1231; Duvoisin et al. (1995), J. Neurosci. 15:3075. As a result, certain neurons may express only one particular mGluR subtype, while other neurons may express multiple subtypes that may be localized to similar and/or different locations on the cell (e.g., postsynaptic dendrites and/or cell bodies versus presynaptic axon terminals). Therefore, the functional consequences of mGluR activation on a given neuron will depend on the particular mGluRs being expressed, the receptors"" affinities for glutamate and the concentrations of glutamate the cell is exposed to, the signal transduction pathways activated by the receptors, and the locations of the receptors on the cell. A further level of complexity may be introduced by multiple interactions between mGluR-expressing neurons in a given brain region. As a result of these complexities, and the lack of subtype-specific mGluR agonists and antagonists, the roles of particular mGluRs in physiological and pathophysiological processes affecting neuronal function are not well defined. Still, work with the available agonists and antagonists has yielded some general insights about the Group I mGluRs as compared to the Group II and Group III mGluRs.
Attempts at elucidating the physiological roles of Group I mGluRs suggest that activation of these receptors elicits neuronal excitation. Various studies have demonstrated that ACPD can produce postsynaptic excitation upon application to neurons in the hippocampus, cerebral cortex, cerebellum, and thalamus as well as other brain regions. Evidence indicates that this excitation is due to direct activation of postsynaptic mGluRs, but it has also been suggested to be mediated by activation of presynaptic mGluRs resulting in increased neurotransmitter release. Baskys (1992), Trends Pharmacol. Sci. 15:92; Schoepp (1994), Neurochem. Int. 24:439; Pin and Duvoisin (1995), Neuropharmacology 34:1. Pharmacological experiments implicate Group I mGluRs as the mediators of this excitation. The effect of ACPD can be reproduced by low concentrations of quisqualate in the presence of iGluR antagonists (Hu and Storm (1991), Brain Res. 568:339; Greene et al. (1992), Eur. J. Pharmacol. 226:279), and two phenylglycine compounds known to activate mGluR1, (S)-3-hydroxyphenylglycine ((S)-3HPG) and (S)-3,5-dihydroxyphenylglycine ((S)-DHPG), also produce the excitation (Watkins and Collingridge (1994), Trends Pharmacol. Sci. 15:333). In addition, the excitation can be blocked by (S)-4-carboxyphenylglycine ((S)-4CPG), (S)-4-carboxy-3-hydroxyphenylglycine ((S)-4C3HPG) and (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG), compounds known to be mGluR1 antagonists. Eaton et al. (1993), Eur. J. Pharmacol. 244:195; Watkins and Collingridge (1994), Trends Pharmacol. Sci. 15:333.
Other studies examining the physiological roles of mGluRs indicate that activation of presynaptic mGluRs can block both excitatory and inhibitory synaptic transmission by inhibiting neurotransmitter release. Pin and Duvoisin (1995), Neuropharmacology 34:1. Presynaptic blockade of excitatory synaptic transmission by ACPD has been observed on neurons in the visual cortex, cerebellum, hippocampus, striatum and amygdala (Pin et al. (1993), Curr. Drugs: Neurodegenerative Disorders 1:111), while similar blockade of inhibitory synaptic transmission has been demonstrated in the striatum and olfactory bulb (Calabresi et al. (1992), Neurosci. Lett. 139:41; Hayashi et al. (1993), Nature 366:687). Multiple pieces of evidence suggest that Group II mGluRs mediate this presynaptic inhibition. Group II mGluRs are strongly coupled to inhibition of adenylyl cyclase, like xcex12-adrenergic and 5HT1A-serotonergic receptors which are known to mediate presynaptic inhibition of neurotransmitter release in other neurons. The inhibitory effects of ACPD can also be mimicked by L-CCG-I and DCG-IV, which are selective agonists at Group II mGluRs. Hayashi et al. (1993), Nature 366:687; Jane et al. (1994), Br. J. Pharmacol. 112:809. Moreover, it has been demonstrated that activation of mGluR2 can strongly inhibit presynaptic, N-type calcium channel activity when the receptor is expressed in sympathetic neurons (Ikeda et al. (1995), Neuron 14:1029), and blockade of these channels is known to inhibit neurotransmitter release. Finally, it has been observed that L-CCG-I, at concentrations selective for Group II mGluRs, inhibits the depolarization-evoked release of 3H-aspartate from rat striatal slices. Lombardi et al. (1993), Br. J. Pharmacol. 110:1407. Evidence for physiological effects of Group II mGluR activation at the postsynaptic level is limited. However, one study suggests that postsynaptic actions of L-CCG-I can inhibit NMDA receptor activation in cultured mesencephalic neurons. Ambrosini et al. (1995), Mol. Pharmacol. 47:1057.
Physiological studies have demonstrated that L-AP4 can also inhibit excitatory synaptic transmission on a variety of CNS neurons. Included are neurons in the cortex, hippocampus, amygdala, olfactory bulb and spinal cord. Koerner and Johnson (1992), Excitatory Amino Acid Receptors; Design of Agonists and Antagonists, p. 308; Pin et al. (1993), Curr. Drugs: Neurodegenerative Disorders 1:111. The accumulated evidence indicates that the inhibition is mediated by activation of presynaptic mGluRs. Since the effects of L-AP4 can be mimicked by L-SOP, and these two agonists are selective for Group III mGluRs, members of this mGluR group are implicated as the mediators of the presynaptic inhibition. Schoepp (1994), Neurochem. Int. 24:439; Pin and Duvoisin (1995), Neuropharmacology 34:1. In olfactory bulb neurons it has been demonstrated that L-AP4 activation of mGluRs inhibits presynaptic calcium currents. Trombley and Westbrook (1992), J. Neurosci. 12:2043. It is therefore likely that the mechanism of presynaptic inhibition produced by activation of Group III mGluRs is similar to that for Group II mGluRs, i.e. blockade of voltage-dependent calcium channels and inhibition of neurotransmitter release. L-AP4 is also known to act postsynaptically to hyperpolarize ON bipolar cells in the retina. It has been suggested that this action may be due to activation of a mGluR, which is coupled to the cGMP phosphodiesterase in these cells. Schoepp (1994), Neurochem. Int. 24:439; Pin and Duvoisin (1995), Neuropharmacology 34:1.
Metabotropic glutamate receptors have been implicated as playing roles in a number of normal processes in the mammalian CNS. Activation of mGluRs has been demonstrated to be a requirement for the induction of hippocampal long-term potentiation and cerebellar long-term depression. Bashir et al. (1993), Nature 363:347; Bortolotto et al. (1994), Nature 368:740; Aiba et al. (1994), Cell 79:365; Aiba et al. (1994), Cell 79:377. A role for mGluR activation in nociception and analgesia has also been demonstrated. Meller et al. (1993), Neuroreport 4:879. In addition, mGluR activation has been suggested to play a modulatory role in a variety of other normal processes including: synaptic transmission, neuronal development, neuronal death, synaptic plasticity, spatial learning, olfactory memory, central control of cardiac activity, waking, motor control, and control of the vestibulo-ocular reflex (for reviews, see Nakanishi (1994), Neuron 13: 1031; Pin and Duvoisin (1995), Neuropharmacology 34:1; Knopfel et al. (1995), J. Med. Chem. 38:1417).
From the forgoing, it will be appreciated that it would be an advancement in the art to identify and characterize novel human metabotropic glutamate receptors and the nucleic acids that code for such receptors. It would be a further advancement to provide methods for screening for agonists, antagonists, and modulatory molecules that act on such receptors.
Such receptors, nucleic acids, and methods are disclosed and claimed herein.
The present invention relates to (1) nucleic acids encoding a newly identified splice variant of human metabotropic glutamate receptor 5 protein and fragments thereof; (2) the metabotropic glutamate receptor protein and fragments thereof; (3) chimeric receptor molecules having one or more domains derived from the new metabotropic glutamate receptor and one or more domains derived from a different receptor; (4) cell lines expressing the metabotropic glutamate receptor protein and fragments thereof; (5) uses of such molecules, nucleic acids, proteins, and cell lines; (6) methods of screening for a compound that binds to or modulates the activity of the metabotropic glutamate receptor; and (7) compounds and methods for modulating the metabotropic glutamate receptor activity and binding to the metabotropic glutamate receptor. Such compounds preferably act as agonists, antagonists, or allosteric modulators of one or more of the metabotropic glutamate receptor activities. By modulating the metabotropic glutamate receptor activities, different effects can be produced, such as anticonvulsant effects, neuroprotectant effects, analgesic effects, psychotropic effects and cognition-enhancement effects.
Metabotropic glutamate receptors have been suggested to play roles in a variety of pathophysiological processes and disease states affecting the CNS. These include stroke, head trauma, anoxic and ischemic injuries, hypoglycemia, epilepsy, anxiety, and neurodegenerative diseases such as Alzheimer""s disease. Schoepp and Conn (1993), Trends Pharmacol. Sci. 14:13; Cunningham et al. (1994), Life Sci. 54:135; Hollman and Heinemann (1994), Ann. Rev. Neurosci. 17:31; Pin and Duvoisin (1995), Neuropharmacology 34:1; Knopfel et al. (1995), J. Med. Chem. 38:1417. Much of the pathology in these conditions is thought to be due to excessive glutamate-induced excitation of CNS neurons. Since Group I mGluRs appear to increase glutamate-mediated neuronal excitation via postsynaptic mechanisms and enhanced presynaptic glutamate release, their activation may contribute to the pathology. Therefore, selective antagonists of these receptors could be therapeutically beneficial, specifically as neuroprotective agents or anticonvulsants. In contrast, since activation of Group II and Group III mGluRs inhibits presynaptic glutamate release and the subsequent excitatory neurotransmission, selective agonists for these receptors might exhibit similar therapeutic utilities. Thus, the various mGluR subtypes may represent novel targets for CNS drug development.
Preliminary studies assessing therapeutic potentials with the available mGluR agonists and antagonists have yielded seemingly contradictory results. For example, it has been reported that application of ACPD onto hippocampal neurons leads to seizures and neuronal damage. Sacaan and Schoepp (1992), Neurosci. Lett. 139:77; Lipparti et al. (1993), Life Sci. 52:85. But, other studies indicate that ACPD can inhibit epileptiform activity (Taschenberger et al. (1992), Neuroreport 3:629; Sheardown (1992), Neuroreport 3:916), and can also exhibit neuroprotective properties (Koh et al. (1991), Proc. Natl. Acad. Sci. USA 88:9431; Chiamulera et al. (1992), Eur. J. Pharmacol. 216:335; Siliprandi et al. (1992), Eur. J. Pharmacol. 219:173; Pizzi et al. (1993), J. Neurochem. 61:683). It is likely that these opposing results are due to ACPD""s lack of selectivity and activation of different mGluR subtypes. A reasonable explanation for the results is that Group I mGluRs were activated in the former studies to enhance excitatory neurotransmission, while the latter effects were mediated by activation of Group II and/or Group III mGluRs to inhibit presynaptic glutamate release, and diminish excitatory neurotransmission. The observations that (S)-4C3HPG, a Group I mGluR antagonist and Group II mGluR agonist, protects against audiogenic seizures in DBA/2 mice (Thomsen et al. (1994), J. Neurochem. 62:2492); while the Group II mGluR selective agonists DCG-IV and L-CCG-I protect neurons from NMDA- and KA-induced toxicity (Bruno et al. (1994), Eur. J. Pharmacol. 256:109; Pizzi et al., J. Neurochem. 61:683) are also consistent with this interpretation.
It is evident that the currently available mGluR agonists and antagonists may be of limited use, both as research tools and potential therapeutic agents, as a result of their lack of potency and selectivity. In addition, since these compounds are for the most part amino acids or amino acid derivatives, they have limited bioavailabilities, which hampers in vivo studies assessing mGluR physiology, pharmacology and therapeutic potential. The identification of agonists and antagonists with a high degree of potency and selectivity for individual mGluR subtypes is therefore the most important requirement to increase the understanding of various mGluRs"" roles in physiological and pathophysiological processes in the mammalian CNS. High-throughput screening of chemical libraries using cells stably transfected with individual, cloned mGluRs may offer a promising approach to identify new lead compounds which are active on the individual receptor subtypes. Knopfel et al. (1995), J. Med. Chem. 38:1417. These lead compounds could serve as templates for extensive chemical modification studies to further improve potency, mGluR subtype selectivity, and important therapeutic characteristics such as bioavailability.
The preferred use of the receptor and methods of the present invention is to screen for compounds which modulate the activity of the novel metabotropic glutamate receptor. However, other uses are also contemplated, including diagnosis and treatment. Such uses are based on the novel metabotropic glutamate receptor identified herein, the amino acid sequence of which is provided in SEQ ID NO: 2, and the DNA coding sequence is provided in SEQ ID NO: 1 (representing the open reading frame (ORF) of human mGluR5d, nucleotides 1-2826).
Thus, in a first aspect, the invention provides a purified or isolated nucleic acid molecule at least 15 nucleotides in length. This nucleic acid codes for at least five contiguous amino acid residues of a unique portion of a metabotropic glutamate receptor protein which has the amino acid sequence provided in SEQ ID NO: 2, a metabotropic glutamate receptor protein which is a contiguous portion of SEQ ID NO: 2, or a functional equivalent of such amino acid sequences. Preferably, the metabotropic glutamate receptor protein is a human protein. In particular embodiments the nucleic acid molecule comprises a genomic DNA sequence, a cDNA sequence, or an RNA sequence. In preferred embodiments, the glutamate receptor protein comprises SEQ ID NO: 2 or a functional equivalent of that sequence. In certain other embodiments, the glutamate receptor protein comprises residues 861 to 942 of the amino acid sequence of SEQ ID NO: 2; these residues form the unique cyotplasmic tail of mGluR5d. Of particular interest are nucleic acid molecules encoding essentially a fill size novel metabotropic glutamate receptor protein. Therefore, in preferred embodiments the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO: 2, or of amino acid residues of 861 to 942 of SEQ ID NO: 2, or of a functional equivalent of those sequences.
It is recognized that a large yet finite number of different nucleic acid sequences will code for the same amino acid sequence due to the redundancy of the genetic code. Such alternative coding sequences are within the scope of the above aspect of the invention.
In a preferred embodiment, the nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2 has the nucleic acid sequence of SEQ ID NO: 1. Also in preferred embodiments, the nucleic acid molecule comprises at least 15 or 50 contiguous nucleotides of the nucleic acid sequence SEQ ID NO: 1 or of a sequence substantially complementary thereto.
Since the use of a modified metabotropic glutamate receptor protein is advantageous in certain applications, in a preferred embodiment, the invention also provides an isolated or purified nucleic acid molecule encoding an amino acid sequence which comprises an extracellular domain which is part of the amino acid sequence of SEQ ID NO: 2. In this embodiment the encoded amino acid sequence is substantially free of membrane spanning domain and intracellular domain portions contained in the amino acid sequence of SEQ ID NO: 2. Likewise, in other particular embodiments, the invention provides other isolated or purified nucleic acid molecules encoding one or more domains which are part of the amino acid sequence of SEQ ID NO: 2, but which do not include at least one such domain. Thus, the invention provides nucleic acid molecules which encode an intracellular domain that is free of transmembrane and extracellular domains, or a transmembrane domain that is free of intracellular and extracellular domains, or an extracellular domain of a metabotropic glutamate receptor that is substantially free of the membrane spanning domains of said metabotropic glutamate receptor, or extracellular and membrane spanning domains which are substantially free of the intracellular domain. Similarly, in particular embodiments, the nucleic acid encodes a metabotropic glutamate receptor that is substantially free of at least one membrane spanning domain portion or a metabotropic glutamate receptor that is substantially free of the extracellular domain of said metabotropic glutamate receptor, or a contiguous multiple-transmembrane domain including intervening intracellular and extracellular domains but substantially free of N-terminal extracellular and C-terminal intracellular domains of SEQ ID NO: 2 (e.g., a seven-transmembrane domain).
In further preferred embodiments the nucleic acid molecule encodes an extracellular domain of SEQ ID NO: 2, transcriptionally coupled to a second nucleic acid molecule which encodes transmembrane and intracellular domains of a protein which is not a metabotropic glutamate receptor protein (i.e., a non-metabotropic glutamate receptor); the purified nucleic acid encodes a fusion protein composed of an N-terminal extracellular domain contiguous with a seven-transmembrane domain of SEQ ID NO: 2 and is transcriptionally coupled to nucleic acid encoding a C-terminal intracellular domain of a non-metabotropic glutamate receptor; the purified nucleic acid encodes a fusion protein composed of an N-terminal extracellular domain contiguous with a seven-transmembrane domain of SEQ ID NO: 2 and is transcriptionally coupled to nucleic acids encoding multiple intracellular domains of a non-metabotropic glutamate receptor.
Since it is advantageous in certain applications to utilize the complementary or anticoding DNA strand, the invention also provides an isolated or purified nucleic acid molecule which has a sequence substantially complementary to the sequence of a nucleic acid molecule of the above aspect.
In the context of this invention, the term xe2x80x9cpurifiedxe2x80x9d means that the specified nucleic acid molecule or polypeptide has been separated from other nucleic acid molecules or polypeptides, respectively, with which it is found in such a manner that it forms a substantial fraction of the total nucleic acids or polypeptides present in a preparation. Preferably, the specified molecule constitutes at least 1, 5, 10, 50, 75, 85, or 95 percent or more of the molecules of that type (nucleic acid or polypeptide) present in a preparation.
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid, polypeptides, or other biomolecules of this invention is meant the molecule is present in a form (i.e., its association with other molecules) other than found in nature. For example, an isolated receptor nucleic acid is separated from one or more nucleic acids which are present on the same chromosome, and an isolated polypeptide is separated from a substantial fraction of the other polypeptides with which it is normally found in nature. Preferably, the isolated nucleic acid or polypeptide is separated from at least 90% of the other nucleic acids present on the same chromosome or polypeptides normally found in the same cell. An example of isolated nucleic acid is recombinant nucleic acid. In this application, the term isolated nucleic acid is distinct from clones existent in a library of clones. It refers to a particular clone having the designated material encoded therein, isolated from other such clones. It can be created by standard recombinant methods to exist within a test-tube or within a desired cell or organism. It is preferably the only nucleic acid cloned within a standard vector, and may or may not contain the naturally occurring control sequences associated with it. Thus, it contains nucleic acid isolated from its natural environment and known to have the sequence claimed to be present. It is preferably a homogenous preparation of nucleic acid separate from other cellular components and from other nucleic acids.
In referring to the nucleic acids and polypeptides of the present invention, the term xe2x80x9cuniquexe2x80x9d refers to a difference in sequence between a nucleic acid molecule of the present invention and the corresponding sequence of other receptor proteins, including other metabotropic glutamate receptor proteins. Thus, the sequences differ by at least one, but preferably a plurality of nucleotides or amino acid residues.
By xe2x80x9csubstantially complementaryxe2x80x9d is meant that the purified nucleic acid can hybridize to the complementary sequence region in a specific nucleic acid under stringent hybridization conditions. Such nucleic acid sequences are particularly useful as hybridization detection probes to detect the presence of nucleic acid encoding a particular receptor. Under stringent hybridization conditions, only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 4 or more mismatches out of 20 contiguous nucleotides, more preferably 2 or more mismatches out of 20 contiguous nucleotides, most preferably one or more mismatch out of 20 contiguous nucleotides. Preferably, the nucleic acid is substantially complementary to at least 15, 20, 27, or 45, contiguous nucleotides of the specific sequence (e.g., in SEQ ID NO: 1).
In the context of the novel receptor and fragments, the term xe2x80x9cfunctional equivalentxe2x80x9d refers to a polypeptide that has an activity that can be substituted for one or more activities of a particular receptor or receptor fragment. This is explained in greater detail in the Detailed Description below.
In reference to the different domains of a metabotropic glutamate receptor, the term xe2x80x9csubstantially freexe2x80x9d refers to the absence of at least most of the particular domain, preferably such that essentially none of an activity of interest specific to that domain remains. Thus, a short portion(s) of the particular domain sequence may remain, but does not provide a substantial particular activity normally provided by the intact domain.
By xe2x80x9ccomprisingxe2x80x9d it is meant including, but not limited to, whatever follows the word xe2x80x9ccomprising.xe2x80x9d Thus use of the term indicates that the listed elements are required, but that other elements are optional and may or may not be present. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant that the listed elements are required, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
Isolated or purified polypeptides corresponding to the nucleic acid molecules of the above aspects are also provided by the present invention. Therefore, in another aspect the invention features a purified polypeptide having at least 6 contiguous amino acids of an amino acid sequence provided in SEQ ID NO: 2. In preferred embodiments, the purified polypeptide has at least 12, 18, or 54 contiguous amino acids of SEQ ID NO: 2. In further preferred embodiments, the purified polypeptide comprises residues 861 to 942 of the amino acid sequence of SEQ ID NO: 2, which form the unique cyotplasmic tail of mGluR5d Other preferred receptor fragments include those having only an extracellular portion, a transmembrane portion, an intracellular portion, and/or a multiple transmembrane portion (e.g., seven transmembrane portion). In a particularly preferred embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
Expression of a recombinant nucleic acid encoding a metabotropic glutamate receptor or receptor fragment is a useful method of producing polypeptides such as those described above. Therefore, in another aspect, the invention provides recombinant nucleic acid encoding a metabotropic glutamate receptor or receptor fragment as described in the first aspect above (i.e., coding for a metabotropic glutamate receptor protein having the amino acid sequence SEQ ID NO: 2 or functional equivalents thereof (i.e., these having one or more of the activities associated with that protein but having a few (1-10) amino acid alterations at non-critical areas which do not affect such activities)), cloned in an expression vector. An expression vector contains the necessary elements for expressing a cloned nucleic acid sequence to produce a polypeptide. An xe2x80x9cexpression vectorxe2x80x9d contains a promoter region (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal protein synthesis initiation. xe2x80x9cExpression vectorxe2x80x9d includes vectors which are capable of expressing DNA sequences contained therein, i.e., the coding sequences are operably linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors must be replicable in the host organisms, either as episomes or as an integral part of the chromosomal DNA. Clearly, a lack of replicability would render them effectively inoperable. A useful, but not a necessary, element of an effective expression vector is a marker-encoding sequencexe2x80x94i.e., a sequence encoding a protein which results in a phenotypic property (e.g., tetracycline resistance) of the cells containing the protein which permits those cells to be readily identified. In sum, xe2x80x9cexpression vectorxe2x80x9d is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified contained DNA code is included in this term, as it is applied to the specified sequence. As such vectors are at present frequently in the form of plasmids, the terms xe2x80x9cplasmidxe2x80x9d and xe2x80x9cexpression vectorxe2x80x9d are often used interchangeably. However, the invention is intended to include such other forms of expression vectors, including viral vectors, which serve equivalent functions and which may, from time to time become known in the art.
In reference to receptor proteins, xe2x80x9cbiologically functionalxe2x80x9d and xe2x80x9cfunctional receptorxe2x80x9d indicate that the receptor molecule or portion has a normal biological activity characteristic of the normal receptor in its usual cellular environment, which is relevant in the process of interest. Such a process can be, for example, a binding assay, or a complex cellular response. Preferably, a functional receptor is capable of participating in the normal cellular response reactions. In reference to an expression vector, xe2x80x9cbiologically functionalxe2x80x9d means that the expression vector can be transcribed and the transcription product translated in the cell or expression system of interest.
The terms xe2x80x9ctransformedxe2x80x9d and xe2x80x9ctransfectedxe2x80x9d refer to the insertion of a foreign genetic material into a prokaroytic or eukaryotic cell. Such insertion is commonly performed using vectors, such as plasmid or viral vectors, but can also include other techniques known to those skilled in the art.
Recombinant nucleic acid may contain nucleic acid encoding a metabotropic glutamate receptor, receptor fragment, or metabotropic glutamate receptor derivative, under the control of its genomic regulatory elements or under the control of exogenous regulatory elements, including an exogenous promoter. By xe2x80x9cexogenousxe2x80x9d is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the metabotropic glutamate receptor.
The expression vector may be used in another aspect of the invention to transform or transfect a prokaryotic or a eukaryotic host cell. Thus, another aspect of the present invention features a recombinant cell or tissue. The recombinant cell or tissue is made up of a recombinant nucleic acid sequence of the first aspect above, and a cell able to express the nucleic acid. Recombinant cells have various uses, including as biological factories to produce polypeptides encoded for by the recombinant nucleic acid, and for producing cells containing a functioning metabotropic glutamate receptor. Cells containing a functioning metabotropic glutamate receptor can be used, for example, to screen for mGluR agonists, antagonists, or allosteric modulators. In preferred embodiments, the cell containing the recombinant nucleic acid encoding a functioning metabotropic glutamate receptor is selected from the group consisting of: central nervous system cell, peripheral nervous system cell, pituitary cell, and hypothalamic cell; and the recombinant nucleic acid encodes at least 12, 18 or 54 contiguous amino acids of SEQ ID NO: 2. In a particular embodiment of the invention the host cell is an oocyte, for example a Xenopus oocyte. In other preferred embodiments, the cell is one of NIH-3T3, HeLa, NG115, CHO, HEK 293 and COS7.
Another aspect of the invention describes a process for the production of a polypeptide product involving growing prokaryotic or eukaryotic host cells transformed or transfected with an expression vector having a nucleic acid molecule which codes for a metabotropic glutamate receptor protein having the amino acid sequence SEQ ID NO: 2, or a portion of that sequence, or a functional equivalent, under suitable nutrient conditions. The host cells are grown in a manner allowing expression of the polypeptide product. In a preferred aspect of the invention the process further involves isolation of the polypeptide product. xe2x80x9cSuitable nutrient conditionsxe2x80x9d are those which will allow a cell to carry on normal metabolic functions and/or grow. The conditions suitable for a particular cell line or strain will generally differ, but appropriate conditions for each such cell type are known to or can be determined by methods known to those skilled in the art.
Another aspect of the invention features a method of screening for a compound that binds to or modulates the activity of a metabotropic glutamate receptor having the sequence SEQ ID NO: 2. The method involves introducing the metabotropic glutamate receptor and a test compound into an acceptable medium and monitoring the binding or modulation by physically detectable means, thereby identifying the compounds which interact with or modulate the activity of the metabotropic glutamate receptor. Such a compound is useful as a therapeutic molecule to modulate metabotropic glutamate receptor activity or as a diagnostic agent to diagnose patients suffering from a disease characterized by an abnormal metabotropic glutamate activity. In a preferred embodiment, the mGluR is a chimeric receptor having an extracellular domain contained in the amino acid sequence of SEQ ID NO: 2 and an intracellular domain of a different receptor. Such a chimeric receptor allows activation of a cellular pathway not normally activated by the novel mGluR described herein. Also, in a preferred embodiment the metabotropic glutamate receptor is expressed by a cell and the compound is screened by monitoring the effect of the compound on the cell. More preferably, the cell is a eukaryotic cell. For example, the method can involve contacting a cell containing a recombinant nucleic acid encoding a metabotropic glutamate receptor with the agent and detecting a change in metabotropic glutamate receptor activity. In another preferred embodiment, the method involves a competition binding assay with a labeled known binding agent. Preferably, the method is used to identify a metabotropic glutamate receptor-modulating agent.
The term xe2x80x9cphysically detectable meansxe2x80x9d refers herein to the means for detecting the interaction between a modulator or binding compound and the novel metabotropic glutamate receptor molecule. Such means can include, for example, spectroscopic methods (e.g., fluorometric measurement of Ca2+), electrophysiological assays, and biochemical assays (e.g., specific enzyme activity). In addition to a variety of other assays, such biochemical assay can include detection of the activation by a chimeric receptor of a cellular pathway not normally activated by the novel mGluR. Each technique detects a physical property or parameter.
A xe2x80x9cchimeric receptorxe2x80x9d is one which has an amino acid sequence which is a fusion or association of sequences from two or more different proteins, at least one of which is a receptor protein. Typically in this invention, a chimeric receptor has amino acid sequences constituting domains (such as extracellular, membrane spanning, and intracellular) from two or more different receptor proteins, one of which is the novel mGluR5d of this invention.
Identification of metabotropic glutamate receptor-modulating agents is facilitated by using a high-throughput screening system. High-throughput screening allows a large number of molecules to be tested. For example, a large number of molecules can be tested individually using rapid automated techniques or in combination with using a combinatorial library of molecules. Individual compounds able to modulate metabotropic glutamate receptor activity present in a combinatorial library can be obtained by purifying and retesting fractions of the combinatorial library. Thus, thousands to millions of molecules can be screened in a short period of time. Active molecules can be used as models to design additional molecules having equivalent or increased activity. Such molecules will generally have a molecular weight of 10,000, preferably less than 1,000.
A further aspect of the present invention describes a method of modulating the activity of a metabotropic glutamate receptor having the amino acid sequence of SEQ ID NO: 2, or a portion, or a functional equivalent, and includes the step of contacting the receptor with a compound that a modulates one or more activities of the metabotropic glutamate receptor, in general either activating or inhibiting activation of the receptor.
The metabotropic glutamate receptor is contacted with a sufficient amount of a compound to modulate a metabotropic glutamate receptor activity. Modulating metabotropic glutamate receptor activity causes an increase or decrease in a cellular response which occurs upon metabotropic glutamate receptor activation, as described in the Detailed Description below. Typically, the compound either mimics one or more effects of glutamate at the metabotropic glutamate receptor, or blocks one or more effects of glutamate at the metabotropic glutamate receptor (or potentially both). The method can be carried out in vitro or in vivo.
The term xe2x80x9cmimicsxe2x80x9d means that the compound causes a similar effect to be exhibited as is exhibited in response to contacting the receptor with glutamate. xe2x80x9cBlocksxe2x80x9d means that the presence of the compound prevents one or more of the normal effects of contacting the receptor with glutamate.
In the context of this invention, xe2x80x9cin vitroxe2x80x9d means that a process is not carried out within or by a living cell(s). However, the process may use cell membranes and other cell parts, or even complete but non-living cells. xe2x80x9cIn vivoxe2x80x9d means that the process is carried out within or by a living cell(s), and thus includes processes carried out within or by complex organisms such as mammals.
With respect to a metabotropic glutamate receptor, xe2x80x9cfunctioningxe2x80x9d or xe2x80x9cfunctionalxe2x80x9d indicates that the receptor has at least some of the relevant biological activities which such a receptor has under normal biological conditions (normal receptor under normal cellular conditions), and preferably substantially all of such activities. These can include, for example, specific binding characteristics and specific enzymatic activity (among others).
Related aspects of the present invention describe agents (e.g., compounds and pharmaceutical compositions) able to bind to the metabotropic glutamate receptor having the amino acid sequence SEQ ID NO: 2, or a portion or functional equivalent thereof. Preferably, the agent can modulate metabotropic glutamate receptor activity.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.